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COLD PCR HRM: a highly sensitive detection method for IDH1 mutations
Author(s) -
Boisselier Blandine,
Marie Yannick,
Labussière Marianne,
Ciccarino Pietro,
Desestret Virginie,
Wang XiaoWei,
Capelle Laurent,
Delattre JeanYves,
Sanson Marc
Publication year - 2010
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.21365
Subject(s) - sanger sequencing , biology , cold pcr , high resolution melt , microbiology and biotechnology , mutant , polymerase chain reaction , genomic dna , dna , rpob , isocitrate dehydrogenase , dna sequencing , gene , genetics , point mutation , 16s ribosomal rna , enzyme , biochemistry
The p.Arg132His mutation of isocitrate dehydrogenase 1 ( IDH1 R132H ) is a frequent alteration and a major prognostic marker in gliomas. However, direct sequencing of highly contaminated tumor samples may fail to detect this mutation. Our objective was to evaluated the sensitivity of a newly described amplification method, coamplification at lower temperature‐PCR (COLD PCR), combined with high‐resolution melting (HRM) for the detection of the IDH1 R132H mutation. To this end, we used serial dilutions of mutant DNA with wild‐type DNA. PCR‐HRM assay detects IDH1 R132H at an abundance of 25%, similar to the detection limit of direct Sanger sequencing. Introducing a run of COLD PCR allows the detection of 2% mutant DNA. Using two consecutive runs of COLD PCR, we detected 0.25% mutant DNA in a background of wild‐type DNA, that mimics a tumor sample highly contaminated by normal DNA. We then analyzed 10 biopsies of tumor edges, considered free of tumor cells by histological analysis, and showed that immunohistochemistry of IDH1 R132H was positive in three cases (30%), whereas double COLD PCR HRM was positive in the 10 cases studied (100%). In summary, COLD PCR HRM analysis is 100‐fold more sensitive than Sanger sequencing, rendering this rapid and powerful strategy particularly useful for samples highly contaminated with normal tissue.Hum Mutat 31:1–6, 2010. © 2010 Wiley‐Liss, Inc.